Regnase-2 inhibits glioblastoma cell proliferation

Regnase-2 (Reg-2/MCPIP2/ZC3H12B) is uniquely expressed at a high level in the healthy brain and down-regulated in samples from patients with glioma, reaching the lowest level in high-grade glioblastoma multiforme (GBM). This RNase is involved in the regulation of neuroinflammation through the degradation of IL-6 and IL-1 mRNAs, key pro-inflammatory cytokines for GBM pathology. Reg-2 is a strong inhibitor of the proliferation of human glioblastoma cell lines and blocks their potential to form colonies. Here, we describe that overexpression of Reg-2 stalls glioblastoma cells in the G1 phase of the cell cycle and reduces the level of transcripts implicated in cell cycle progression. These newly identified targets include CCND1, CCNE1, CCNE2, CCNA2, CCNB1, and CCNB2, encoding the cyclins as well as AURKA and PLK1, encoding two important mitosis regulators. By RNA immunoprecipitation we confirmed the direct interaction of Reg-2 with the investigated transcripts. We also tested mRNA regions involved in their interaction with Reg-2 on the example of CCNE2. Reg-2 interacts with the 3’UTR of CCNE2 in a dose-dependent manner. In conclusion, our results indicate that Reg-2 controls key elements in GBM biology by restricting neuroinflammation and inhibiting cancer cell proliferation.

structure present in the 3'UTR of interleukin-6 (IL-6) mRNA Reg-2 restricts its level 6,7 .So far, only a few other targets for Reg-2 have been described.They include mRNAs coding another pro-inflammatory cytokine interleukin-1 (IL-1), apoptotic factor IER-3, and a member of the Regnase/MCPIP/ZC3H12 family RNase Reg-1 6,7 .Importantly, both interleukins identified so far as Reg-2 targets are well-described crucial players in GBM biology.Using the TCGA database and patient-derived primary glioma cells, it was established that the expression of IL-6 or IL-1 (and their receptors) is a predictor of an unfavorable prognosis 5,11 .Therefore Reg-2 controls the turnover of transcripts of the cytokines which are significantly correlated with poor glioma patient survival.Recently we have discovered that Reg-2 inhibits the proliferation of human glioblastoma cell line U251-MG and mouse glioblastoma cell line KMWT1 established from the spontaneous glioma tumor 7 .Despite the significant effects of Reg-2 on glioma cells the proliferation mechanisms by which this phenotype is reached remain elusive.
Here we reveal the details of Reg-2-dependent inhibition of cell proliferation.

Reg-2 inhibits the proliferation of human glioblastoma cell line U87-MG
We have already shown that Reg-2 restricts the proliferation of the U251-MG human glioblastoma cell line and KMWT1 mouse spontaneous glioblastoma cells 7 .To confirm that this phenomenon is universal we analyzed the influence of Reg-2 on another human glioblastoma cell line derived from a malignant glioblastoma tumor, U87-MG.Using the Sleeping Beauty (SB) transposon system we generated U87-MG cell lines with introduced sequences encoding WT Reg-2 (U87-Reg-2) or its mutein Reg-2-D/A (U87-Reg-2D/A) or luciferase (U87-luc) under the control of doxycycline (dox)-inducible promoter.Reg-2-D/A is a nucleolytically inactive protein due to the replacement of one of the crucial aspartic acids in the NYN/PIN domain, required for the binding of magnesium ions, by alanine (D196A) 6,7 .Without dox stimulation, all modified cells divide at the same rate.However, as shown in Fig. 1a the time-dependent increase in the cell number is blocked when WT Reg-2 is expressed.The number of U87-Reg-2 cells remains almost constant within 72 h of their culture in a medium containing dox.Simultaneously, the proliferation of control U87-luc cells under the same conditions remains exponential within the examined period with a doubling time of approx.24 h.The observed effect of Reg-2 on cell proliferation mainly relies on its nucleolytic activity; however, the number of U87-Reg-2D/A cells after 72 h of dox stimulation is slightly reduced in comparison to untreated and control cells.
Since the detected effect of Reg-2 on cell number could be due to cell death and/or inhibition of their proliferation we employed the CellTrace Far Red assay to monitor cell proliferation.Cells were stained with CellTrace Far Red reagent (Thermo Fisher Scientific) and cultured for 48 h with/without dox.Cell division was tracked by measuring the fluorescent signal generated after the conversion of cell permeable fluorescent molecules to their derivatives by intracellular esterases.As a result of cell division and the spread of fluorescent dye, the signal decreases approximately two-fold per division.Figure 1b shows the shift toward higher intensity of the peak in U87-Reg-2 cells treated with dox.Such an outcome suggests that induction of Reg-2 overexpression results in slower proliferation of U87-Reg-2 cells.The effect of ribonucleolytic inactive mutein (Reg-2-D/A) on U87 cell proliferation is much weaker (Fig. 1b).
To further determine the influence of Reg-2 on cell division we performed a clonogenic assay.This tool allows monitoring in vitro the ability of single cells to grow into a colony.Reg-2 overexpression strongly inhibits glioma cell division and its potential to form colonies (Fig. 1c).This regulation requires an intact NYN/PIN domain as the effect of nucleolytic-inactive Reg-2-D/A on colony formation was similar as in the control cells (Fig. 1c).

Impact of Reg-2 on the glioblastoma cell cycle
To reveal the mechanisms responsible for Reg-2-dependent inhibition of proliferation, we analyzed its influence on the cell cycle.U87-Reg-2, U87-Reg-2D/A, and control cells were stained with propidium iodide dye, and the number of cells in the individual phases of the cell cycle was estimated by flow cytometry.After dox induction, the population in phase G1 increases from ~ 70% in control to ~ 80% in U87-Reg-2 cells.A similar but much weaker trend is observed in U87-Reg-2D/A cells.The S phase is not significantly affected by Reg-2 overexpression in investigated cells.Cells in the G2 phase accounted for ~ 11% in U87-Reg-2, ~ 13% in U87-Reg-2D/A, and ~ 16% in control cells (Fig. 1d).We concluded that Reg-2 stalls U87-MG glioblastoma cells in the G1 phase.This process relies mainly on its nucleolytic activity; however, inactive ribonucleolytic Reg-2 also exerts a weak effect.

Reg-2 targets are involved in cell cycle transitions and are up-regulated in samples from patients with glioblastoma
To identify mRNA targets implicated in cell cycle regulation, we have performed an RNAseq analysis of the transcriptome of U251-MG human glioblastoma cells overexpressing Reg-2 or luciferase (control).Obtained results have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE248634.Of the 29 analyzed transcripts encoding proteins involved in the regulation of proliferation, 21 mRNAs encoding cyclins, kinases, and regulatory proteins were significantly down-regulated by Reg-2 (Table 1).Since our results indicate the involvement of Reg-2 in cell cycle control during the G1/S transition, we have selected the CCND1, CCNE1, CCNE2, and CCNA2 mRNAs (encoding cyclins D1, E1, E2, A2 respectively) for further analysis 12 .Interestingly, among transcripts affected by Reg-2, there are also mRNAs encoding proteins required for G2/M phases of the cell cycle 12 .To verify these data we have chosen CCNB1 (encoding cyclin B1), and CCNB2 (encoding cyclin B2) for additional examination.We have also selected two additional transcripts encoding kinases important for cell cycle progression: PLK1 encoding Polo-like kinase 1, a critical regulator of cell cycle progression, and AURKA encoding Aurora kinase A involved in different aspects of mitotic control.During cell cycle progression the functional cross-talk between these two kinases exists and both are important players in glioblastoma development 13,14 .Low levels of their mRNAs have been shown to have antiproliferative  effects in glioma 15 .Results obtained by qPCR in U87-Reg- www.nature.com/scientificreports/(Fig. 2a).As a positive control in this analysis, we have used a well-known Reg-2 target, mRNA for IL-6.Consistent with previously published results, Reg-2 regulates IL-6 mRNA in a NYN/PIN-dependent manner (Fig. 2a).
To study the mechanisms of observed changes in transcript levels we have employed RNA immunoprecipitation (RIP).Cells were modified by SB transposon enabling the expression of Reg-2-D/A with N-terminal HALO-Tag and TwinStrep Tag.We have chosen Reg-2-D/A for these experiments to avoid target degradation.PCR analysis of immunoprecipitated mRNAs reveals direct binding of Reg-2 to all tested transcripts.As a positive control, we have used IL-6 mRNA and, as a negative control, POLE4 mRNA.Reg-2 is bound to the IL-6 transcript and does not interact with POLE4 mRNA (Fig. 2b).
To confirm that the observed regulation depends on the interaction of Reg-2 with the 3'UTRs of the transcripts, we have cloned the 3'UTR of CCNE2 into a luciferase reporter vector.The co-expression of luciferase mRNA with attached CCNE2 3'UTR and the expression vector encoding Reg-2 results in decreased luciferase activity in transfected cells.Reg-2 overexpression correlates with a lower luciferase activity in a dose-dependent manner.The regulation requires the presence of CCNE2 3'UTR, as no decrease in luciferase activity is observed when this region is absent (Fig. 2c).
We have analyzed the level of Reg-2 and its newly identified targets that encode the cyclins CCND1, CCNE1, CCNE2, CCNB1, CCNA2, and the kinases PLK1 and AURKA in glioma tumors.The quantity of Reg-2 shows a correlation with the tumor grade, being at its lowest level in GBM.Conversely, higher mean expression levels for all investigated Reg-2 targets were noted in GBM samples compared to LGG samples.However, due to the high level of heterogeneity of GBM samples, statistical analysis of the examined sample group acknowledged significant changes only for CCNB1, CCNA2, PLK1, and AURKA, not for CCND1, CCNE1, and CCNE2 (Fig. 3a).Nonetheless the data from publicly available databases confirm that the level of all investigated transcripts changes substantially between healthy and tumor tissue (Fig. 3b).

Changes in Reg-2 targets are detected also on the protein level
To confirm that the presence of Reg-2 correlates with decreased levels of proteins encoded by the newly discovered Reg-2 substrates, we have employed Western Blot analysis.In U87-Reg-2 cells overexpressing wild-type Reg-2 the reduction in the level of cyclin D1, E1, E2, A2, B1, and Aurora kinase A is observed.Of these proteins, cyclin D1, E2, and A2 are also decreased in U87-Reg-2D/A cells, confirming the NYN/PIN-independent mechanism of their regulation by Reg-2 (Fig. 4).We have performed a similar analysis in another glioblastoma cell line, U251-MG.The cells were modified using the SB system containing the coding sequence for Reg-2 (U251-Reg-2), Reg-2-D/A (U251-Reg-2D/A) or luciferase (control) under the dox-inducible promoter.The obtained results reveal a similar pattern of the regulation of the investigated proteins.Cyclins D1, E1, E2, B1, and Aurora kinase A are down-regulated in U251-Reg-2 but cyclins D1, E2, and A2 are also decreased in U251-Reg-2D/A cells.(Fig. 4).Obtained results show that changes in the amount of investigated proteins reflect changes detectable on the mRNA level.

Discussion
All Reg/ZC3H12/MCPIP family members of RNases are involved in transcript turnover.Their specificity partly overlaps, for example, IL-6 mRNA is degraded by all family members.However, it is hard to determine the extent of the overlapping substrates as our knowledge about this family's targets is mainly limited to identified Reg-1 targets.So far, merely a few Reg-2 substrates have been identified, namely IL-6, IL-1, IER3, and Reg-1 mRNAs.In this report, we demonstrate that overexpression of Reg-2 in human glioblastoma cell lines down-regulates the level of several transcripts involved in the regulation of cell cycle progression such as CCND1, CCNE1, CCNE2, CCNA2, CCNB1, CCNB2, PLK1, and AURKA.By RIP we have confirmed that this regulation takes place through the direct interaction of Reg-2 with investigated transcripts.The amount of Reg-2 is reduced in samples from patients with GBM, thus the decrease in its level may be one of the mechanisms responsible for disturbances in cell cycle advancement.Abnormal activity of the cell cycle machinery is detected in all tumors and is the foundation of tumorigenesis.Well-documented changes in cell homeostasis linked to the control of DNA replication and mitosis include changes in gene expression, protein abundance, and protein post-translational modification.Recently, changes in transcript turnover have also been described as an important mechanism involved in the control of cell division; however, our knowledge about this aspect is very limited.So far, only two RNases participating in this process have been identified: ribonuclease mitochondrial RNA processing (MRP) and Reg-1.MRP is involved in cell cycle regulation and mitochondrial DNA replication 16 .This RNase cleaves CLB2 mRNA in its 5'UTR, followed by its degradation by Xrn1 exonuclease.Disturbances in MRP activity caused by mutations lead to an increased level of CLB2 and the encoded protein Clb2p (B-cyclin).As a result, late anaphase is delayed 17 .Mutation in the MRP subunit causes a recessively inherited developmental disorder, cartilage-hair hypoplasia 18 .Reg-1 overexpression restricts the level of CCND1, CCND3, CCNE1, CCNE2, CDC2, AURKA, and MCM10 transcripts in the MV3 human melanoma cell line 19 .In human neuroblastoma cell lines, KELLY and BE(2)-C, Reg-1 down-regulates CCND3, CCNE1, CCNE2, CCNA2, and CCNB1 mRNA 20 .In samples from patients with melanoma or neuroblastoma, the reduction of Reg-1 level has been detected, thus abnormalities in cell cycle progression in these tumors can result from the down-regulation of Reg-1 19,21 .
Glioblastoma cells that exhibit increased levels of Reg-2 undergo a reduction in their uncontrolled cell division potential.Elevated expression of Reg-2 results in the suppression of both their proliferation and their ability to form colonies (Fig. 1).This phenomenon is predominantly dependent on the RNase activity of Reg-2, as the overexpression of inactive/mutated form of Reg-2 only marginally impedes proliferation, without any noticeable effect on colony formation.Reg-2 overexpression stalls glioblastoma cells in the G1 phase, and among its newly identified targets we have detected mRNAs for cyclin D1, and cyclins E1 and E2 that act in the early and late G1/S phase respectively.However, Reg-2 also regulates the level of transcripts of cyclins that predominate in other cell cycle phases such as cyclin A2, B1, and B2 (phase G2/M).Furthermore, Reg-2 restricts the levels of PLK1 and AURKA mRNAs encoding regulatory serine/threonine kinases involved in mitotic entry in an NYN/PIN-dependent manner.These kinases are in complex interplay through the cell cycle progression, with Aurora kinase A involved in the phosphorylation-mediated activation of PLK1 13 .Both kinases are overexpressed in glioma, and their high level is correlated with a worse prognosis in patients.Both are also considered to be potential targets for glioblastoma therapy 22,23 .Down-regulation of their mRNA level is correlated with an antiproliferative effect 15 .Some of the identified Reg-2 targets, CCND1, CCNE2, and CCNA2 are down-regulated independently of the presence of its active endonucleolytic domain, although the effect of wild-type Reg-2 is more profound than that of inactive mutein.The impact of mutein on the transcript level suggests a possible role of Reg-2 in the recruitment of other nucleases to Reg-2 targets.So far, the interaction of Reg-1 and Reg-4 on IL-6 mRNA has been described 24 .Hence, a plausible explanation for the observed impact of Reg-2D/A on the levels of CCND1, CCNE2, and CCNA2 mRNAs could involve the interaction of Reg-2 with other enzymes acting on these transcripts.The proteins from the Reg/MCPIP/ZC3H12 family recognize stem-loop structures located in the 3'UTR of their targets 6,25 .We have chosen one of the newly identified Reg-2 substrates, CCNE2, to study this issue.CCNE2 3'UTR cloned into the reporter vector changes the stability of reporter mRNA.Reg-2 overexpression negatively regulates the level of the reporter protein translated on the mRNA containing CCNE2 3'UTR.This regulation is dose-dependent.
In this paper, we emphasize the existence of a novel way of regulating the cell cycle.The RNases involved in transcript turnover accompaniment well-known elements such as transcription factors regulating the expression of genes encoding proteins engaged in the cell cycle and machinery responsible for their posttranslational modifications and degradation.
Our results indicate a key role of Reg-2 in glioblastoma biology.Through the regulation of neuroinflammation and restriction of proliferation, Reg-2 controls crucial elements of glioblastoma development.

Transfection, stimulation, and reporter gene assay
U251-MG cells were seeded in 24-well plates at a density of 5 × 10 4 cells/well.The following day, the cells were transfected with PEI reagent (Polysciences, USA) according to the manufacturer's instructions.A total amount of 800 ng of plasmid DNA per well was used, including 400 ng of the luciferase-coding reporter plasmid (pmirGLO or pmirGLO-CCNE2-3′UTR) and varying amounts of Reg-2 expression vector.The quantity of DNA/well was normalized using an empty pcDNA3.1/mycHisAvector (Invitrogen, USA).24 h after transfection cells were lysed and firefly and Renilla luciferase activity were measured using Dual-Luciferase Reporter Assay System (Promega, USA), according to the manufacturer's instructions.For transfection efficiency normalization the Renilla luciferase, encoded on the same plasmid as the firefly luciferase (pmirGLO), was used.

RNA immunoprecipitation
HeLa cells with Dox-inducible expression of Reg-2D/A N-terminally tagged with HALO and TwinStrep Tags separated by HRV 3C cleavage site or luciferase (control cells, transfected with pSB-tet-Pur plasmid) were seeded on 150-mm cell culture dishes and the following day transgene expression was induced with 1 µg/ml Dox.After 24 h the cells were UV-irradiated at 254 nm with 400 mJ/cm 2 , lysed in modified RIPA buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1% Triton-X-100, 0.25% sodium deoxycholate, 3 mM MgCl 2 , 5 mM DTT, 1 × Protease Inhibitor Cocktail (Promega) and 1U/ml RNase Inhibitor (A&A Biotechnology) and homogenized using a syringe and a 23G needle.After homogenization cell debris was removed by centrifugation, 1/10 of the supernatants were kept as input samples, and the rest was used for two-step purification.In the first step the lysate was diluted 5 × in HaloTag® protein purification buffer (50 mM HEPES pH 7.

RNAseq
To identify new targets of Reg-2 we performed RNAseq analysis.Using the SB transposon system we have modified U251-MG cells to generate a cell line with Dox-inducible expression of Reg2, or luciferase (control cells).
To identify differentially expressed genes we extracted total RNA from control and Reg-2 overexpressing cells (using Zymo Research Direct-zol RNA MiniPrep kit, cat.R2053) and sent samples (in tryplicates) for complete analysis.Assessment of RNA quality, library preparation, RNA seq, and all data analysis was performed by Qiagen Genomic Services, Germany.

Cell count/clonogenic assay
For clonogenic assay cells were seeded in triplicates at a density of 100 cells per well on 6-well plates.Doxycycline (1 µg/ml) was added daily to half of the wells to induce the expression of the transgene.When visible colonies appeared (after ~ 10 days) cells were fixed using a Clonogenic assay fixer/stain (6% vol/vol glutaraldehyde, 0.5% wt/vol crystal violet), and whole plates were scanned using the Olympus IX83 microscope.Quantitative analysis was performed by determining the percentage of area covered by crystal violet stained colonies using ImageJ software.
For cell count assay U87-MG-Reg-2/Reg-2 D/A and control (transfected with luciferase-coding vector) cells were seeded in 6-well plates at the density of 80,000 cells per well.To induce transgene expression, doxycycline (1 µg/ml) was added every 24 h.Cells were trypsinized and counted after 24, 48, and 72 h from expression induction using the TC-20 cell counter (BioRad).

RNA isolation and reverse transcription
Tissue sample collection was performed following Jagiellonian University Ethics Committee guidelines and regulations.Informed consent was obtained from all subjects and/or their legal guardian(s).Before RNA isolation tissue samples were preserved in Nucleic Acid Preservation buffer (prepared according to M. Camacho-Sanchez et.al. 2013) 30 and stored at − 80 °C.The samples were then homogenized in phenozol [1:1 phenol: GTC (4 M guanidinium thiocyanate, 25 nM sodium citrate, pH 7.0, 0.05% (w/v) sarkosyl and 0.1 M 2-mercaptoethanol)], and total RNA isolation was performed according to the Chomczynski modified protocol.Cells were lysed in GTC buffer and RNA isolation was performed according to Chomczynski's protocol.RNA quantification and purity were assessed using spectrophotometric measurements using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific).RNA integrity was assessed by denaturing, formaldehyde gel electrophoresis.The reverse transcription reaction was carried out using 1 µg of total RNA with M-MLV-Reverse transcriptase (Promega) and 500 ng of random hexamer primers (EurX) according to the manufacturer's instructions.The cDNA was used in quantitative real-time PCR (qRT-PCR) for the evaluation of the amounts of the mRNAs of interest.

Figure 1 .
Figure 1.Regnase-2 inhibits proliferation, and stalls the cell cycle at the G1 phase.(a) U87-MG cells were treated with dox (1 μg/mL) or left untreated and counted 24, 48, and 72 h after the addition of dox.The levels of Reg-2 and Reg-2 D/A were examined using western blotting.The graph shows results from three independent experiments + / − SD.(b) U87-luc, Reg-2, or Reg-2D/A cells were labeled using CellTrace Far Red reagent and divided into two dishes.One dish was induced with doxycycline (1 μg/ml), the other was left untreated.After 48 h of induction, the fluorescence was measured by flow cytometry.The graphs show histogram overlays of noninduced (gray) and induced (white) cells.Representative results of three independent experiments are shown.(c) U87-MG cells were plated on 6-well plates (100 cells/well), and treated with dox (1 μg/ml) daily until visible colonies were formed (10 days).Then the colonies were fixed and stained.Graph presents the ratio of the surface covered by colonies formed by dox-induced to non-induced cells normalized to control cells (luc) (mean value of three independent experiments ± SD) (d) U87-luc/Reg-2/Reg-2D/A cells were treated with dox (1 µg/mL) for 48 h, and cell cycle analysis was performed.The graphs show a histogram of propidium iodide fluorescence in particular cell lines.Representative results of three independent experiments are shown.The graph and table show the percentages of cells in each cell cycle phase.The data were analyzed using two-way ANOVA and Bonferroni's post-hoc test (***p < 0.001; *p < 0.05).

Figure 2 .
Figure 2. Reg-2 regulates the level of transcripts down-regulated in glioblastoma samples and involved in cell cycle regulation.(a) U87-MG cells with sleeping beauty (SB) transposon-based dox-inducible expression of luciferase (luc), wild-type Reg-2 (Reg-2), or mutein Reg-2 D196A (Reg-2D/A) were treated with dox (1 μg/ml) or left untreated for 24 h, and the levels of the indicated mRNAs were examined using qRT-PCR.Graphs show mean results presented as normalized values (induced/noninduced) from four independent experiments + / − SD.Data were analyzed using two-way ANOVA with Bonferroni's post-hoc test (*p < 0.05; **p < 0.01; ***p < 0.001).(b) Reg-2 directly binds endogenous mRNA encoding tested proteins.HeLa cells were transfected with vectors encoding the Reg-2-D/A mutein (with N-terminal HALO-Tag and TwinStrep Tag)or a luciferase-coding control vector, and after UV-crosslinking, the RNA immunoprecipitation (RIP) was performed.The immunoprecipitated RNA as well as RNA isolated from input samples were used for reverse transcription and qRT-PCR.Next, qRT-PCR products were run on 2% agarose gel with Ultra Low Range DNA Ladder (Thermo Fisher Scientific).In case of CCND1, increased contrast image with pseudocolor mask for visualization of low intensity bands is also presented.Image was prepared using ImageJ software.(c) Reg-2 is involved in the 3′UTR-dependent destabilization of CCNE2 transcript.U251-MG cells were transfected with vectors encoding luciferase with the attached 3′UTR of CCNE2 or without any additional 3′UTR (pmirGLO) and an expression vector for Reg-2 (80 ng or 160 ng).The graphs show the mean results of three independent experiments + /− SD.The data were analyzed using two-way ANOVA and Bonferroni's post hoc test (***p < 0.001).

Figure 3 .
Figure 3. (a) The level of Reg-2, CCND1, CCNE1, CCNE2, CCNA2, CCNB1, AURKA, and PLK1 in clinical samples of low-grade glioma (LGG) and glioblastoma multiforme (GBM).Tumor samples were homogenized and subjected to RNA isolation.Each point represents a single patient.The levels of the indicated mRNAs were examined using qRT-PCR.The horizontal lines represent the mean + / − SEM.The data were analyzed using the Mann-Whitney test.(*p < 0.05; **p < 0.01).(b) Expression of selected transcripts in normal and cancer tissues of the human brain.Numbers in parentheses indicate the number of samples and data sets, respectively.The red line indicates the average, the black line the median, the box upper side the Q3 percentile and the lower side the Q1 percentile, and the upper and lower error bars indicate the maximum and minimum respectively.Graph prepared from the data obtained from the Oncopression database.

Figure 4 .
Figure 4. Reg-2 downregulates the level of proteins involved in cell cycle regulation.U87-MG or U251-MG with Sleeping Beauty (SB) transposon-based dox-inducible expression of luciferase (luc), wild-type Reg-2 (Reg-2) or mutein Reg-2 D196A (Reg-2 D/A) were treated with dox (1 μg/mL) for 24 h, then protein lysates were collected, and analyzed by western blotting.A representative image of two to three independent experiments with similar results is shown.Values below lanes show densitometry analysis (ratio of analyzed protein to total protein was normalized to control sample and presented as mean ± SD).

domain Category Gene Description ENSEMBL IDENTIFIER Reg-2 vs ctrl fold change p-value FDR p-value CYCLINS CCNE2 Essential for the control of the cell cycle at the late G1 and early S phase ENSG00000175305 − 2.11 0 0 CCNE1 Essential for the control of the cell cycle at the G1/S transition ENSG00000105173 − 1.43 6.19E-06 8.74E-05 CCND1 Regulates the cell-cycle during G1/S transition
2 and U87-Reg-2D/A indicate that the levels of CCNE1, CCNB1, CCNB2, PLK-1, and AURKA are restricted by Reg-2 in an NYN/PIN-dependent manner.The levels of CCND1, CCNE2, and CCNA2 are down-regulated by Reg-2 independently of the active nucleolytic

CCNB1 Essential for the control of the cell cycle at the G2/M transition ENSG00000134057 − 1.21 7.40E-03 0.05 CCNB2 Essential for the control of the cell cycle at the G2/M transition ENSG00000157456 − 1.21 7.12E-03 0.04 CCNA2 Controls both the G1/S and the G2/M transition phases of the cell cycle ENSG00000145386 − 1.26 9.99E-04 8.13-03
CDK1Catalytic subunit of the kinase complex known as M-phase promoting factor (MPF), which is essential for G1/S and G2/M phase transitions of cell cycle ENSG00000185324 − 1.03 0.66 0.82 PLK1 Critical regulator of cell cycle progression, mitosis, cytokinesis, and the DNA damage response ENSG00000166851 − 1.37 1.81E-05 2.28E-04

Table 1 .
Differential expression of selected mRNAs involved in proliferation and cell cycle regulation in Reg- 2-overexpressing vs control U251-MG cells based on RNAseq analysis.Table shows fold change of selected transcripts together with p-value and FDR-adjusted p-value.Transcripts selected for further analysis are bold.Vol.:(0123456789) Scientific Reports | (2024) 14:1574 | https://doi.org/10.1038/s41598-024-51809-x Cells modified with SB transposons were seeded in 12-well plates.24 h after transfection, protein expression was induced by adding doxycycline (1 µg/ml).The following day, cells were lysed in SDS loading buffer (0.35 M Tris•HCl, 35% (v/v) glycerol, 10% (w/v) SDS, 3.6 M β-mercaptoethanol, 0.12 g/ml bromophenol blue) and denatured at 95 °C for 7 min.Protein extracts were separated on SDS-PAGE electrophoresis, transferred to low fluorescence PVDF membrane (Millipore), and analyzed by Western blotting.For total protein UV-visualization 2,2,2-trichloroethanol (TCE) was added to polyacrylamide gels to a final concentration of 0.5% v/v 29 .Fx documentation system (Vilber Lourmat).Images were cropped and prepared using Quantity One software.All modifications (brightness and contrast adjustments) were applied equally across the entire image and applied equally to controls.Figures containing Western Blot images were prepared using Inkscape software.Original uncropped images with visible edges, as well as images combined with protein ladder are provided in Supplementary Informations.The densitometry analysis was performed on raw images using ImageJ software.
× Buffer BXT (IBA Lifesciences) containing 200 mM d-biotin.The pooled eluates were mixed 1:1 with 2 × proteinase K digestion buffer (20 mM Tris-HCl pH 8.0, 200 mM NaCl, 2 mM EDTA, 1% SDS) and digested for 0.5 h at 50 °C with 400 µmg/ml proteinase K (Ambion) along with the input samples.After digestion the RNA was purified by phenol/chloroform extraction and isopropanol precipitation with Pellet Paint NF (Merck) used as a coprecipitant.The purified RNA was reverse transcribed to cDNA using MMLV Reverse transcriptase (Promega) and a 1:1 mix of oligo(dT)15and random hexamer primers and subjected to semiquantitative PCR for evaluation of the Reg-2 bound transcripts.